The production of a recombinant biotechnological or biopharmaceutical product (protein, virus, RNA, DNA, antibody, coagulation factor, enzyme) involves not only the expression of the target substance in cell cultures in fermenters, which is also referred to as upstream processing (USP), but also the purification of the target substance from contaminants found together in solution (host cell proteins, DNA, RNA, media constituents, undesired viruses, etc.). The latter step is also referred to as downstream processing (DSP). The central step in DSP is the chromatographic separation of the substances situated in the inflow, the feedstream. This involves the binding of certain substances to a solid matrix, which can be a membrane, a particulate gel or a monolith. Fundamentally, chromatographic steps in DSP are carried out in two configurations. Depending on whether the target substance or the target molecule binds to the chromatographic matrix and is then eluted, or resides in the mobile phase and undesired contaminants bind to the matrix, said configurations are referred to as bind-and-elute or as flow-through applications.
The binding capacity of chromatographic matrices both for target molecules or contaminants is limited and the costs can be very high, for example for antibody purification using affinity matrices. Consequently, from the perspective of process economy, a favorable situation ensues when chromatographic matrices are dimensioned as small as possible and the available binding capacity is utilized as fully as possible. This is limited by the fact that there should be avoidance of a loss due to overloading of a column in bind-and-elute mode or of an input of contamination due to an overloading of the chromatographic matrix in flow-through mode. The loading status of a chromatographic matrix can be monitored by means of a sensor, such as, for example, a UV or IR sensor, which is situated at the output side. If the capacity of a matrix is exceeded, this results in a change in the signal intensity at the output of the medium. The attainment of a predefined value can be used as a termination criterion, at which the loading of the column is stopped. An important parameter in this method is the signal-to-noise ratio of customary detectors used in processes, and this can have a direct effect on the losses of target molecule (bind and elute) or on the contamination of the feedstream with secondary components (flow-through).
Therefore and owing to (i) batch-dependent variations in the binding capacity of the chromatographic media, (ii) errors in the case of scaled enlargement of established processes, and (iii) the need to take possible extreme cases into account, for example for variations in the concentration of the molecule in the feedstream, but also in pH and in conductivity, the theoretically available capacity of a chromatographic matrix is in practice not fully utilized in order to be safe. Generally, the utilization of the capacity of the chromatographic matrix is estimated to be only approx. 60%. Although the loss of target molecule or the contamination of the process stream can be avoided in this way, the overdimensioning of the chromatographic matrix leads to an economically unfavorable situation. An optimization of the utilization of the capacity of the chromatographic matrix can therefore provide a large savings potential in process time and in material costs, especially in the case of cost-intensive chromatographic materials such as protein A.
WO 2010/083859 A1 discloses a device and a method for isolating substances from a mixture, the device comprising at least one diffusively operable and one convectively operable chromatography matrix. In the examples mentioned, the connection of the diffusively operable matrix (column) to a smaller convectively operable matrix (membrane adsorber) leads to an improved, i.e., steeper, breakthrough behavior during loading and a twice as high productivity (mg/ml×min). Owing to the combination of a column with a membrane adsorber, it is possible to increase the efficiency of the separation process and to improve the breakthrough behavior of the separation process.
WO 2010/151214 A1 discloses a system which captures the loading status of a chromatographic column, by capturing the input signal of a column via a first detector and also the output signal via a second detector. The ratio of the input signal and the output signal yields conclusions with regard to the loading status of the column. This information can be used in the context of a multistep chromatographic process in order to define the start and stop of different process steps, such as loading for example. The device disclosed in WO 2010/151214 A1 makes it possible to determine binding capacities of chromatographic columns and fundamentally makes use of two sensors in order to ascertain the loading status of a column.
WO 99/34220 A2 discloses a method which can capture via an online detection method the loading status of a chromatographic unit containing a solution consisting of a target molecule and contaminants. In said method, a small portion of the eluate of the chromatographic unit is deflected from the eluate stream into a separate detection unit. The separate detection unit, which can, for example, be a chromatographic structure, ascertains the proportion of contaminants and unbound target molecule in less than 20% of the total elution time. Loading can be terminated when a defined concentration of target molecule in the eluate is exceeded. In this method, it is necessary to divert a portion of the eluate of a chromatographic column into a separate detection unit. A return of the proportion of the eluate which was used for the analysis back into the eluate stream is not envisaged or is made impossible by the chromatographic steps during the analysis and the solvents used.
U.S. Pat. No. 7,901,581 B2 discloses a system composed of at least three chromatography matrices which are connected to one another by valves and can be continuously operated as a “simulated moving bed” concept. The system is controlled by means of a control unit and refers to UV measurement values of the eluates of the individual units. One function of the structure is the catching of unbound target molecule from the loading solution or wash fraction of a first column by a downstream column, with at least three separately controllable chromatographic matrices being required. It is therefore possible to prevent unbound target molecule from being lost from the process.
EP 1 718 668 B1 discloses a method for purifying antibodies from a solution containing contaminants. In said method, said solution is contacted with a chromatographic matrix on which multimodal ligands have been immobilized. In this connection, the multimodal ligands comprise at least one cation-exchanging group and one aromatic or heteroaromatic ring system. The chromatographic matrix can be particulate, a monolith or a membrane. In this connection, the antibody-containing solution used for loading the matrix is an eluate from an affinity-chromatographic step such as protein A affinity chromatography. Mentioned as possible operating modes of the multimodal chromatography matrix are both the flow-through application, in which only contaminants and, inter alia, also protein A adsorb, and the bind-and-elute application, in which the antibodies and the contaminants adsorb and can then be eluted separately. A switch between the two operating modes can be made by adjusting the pH of the solution after the affinity-chromatographic step. The disclosure mentioned here describes the use of a multimodal chromatography matrix alone or in a two-column arrangement following an affinity-chromatographic step with the goal of a maximally economical purification of antibodies. In the case of the two-column arrangement, the advantages of the combination of two different matrices (affinity chromatography and multimodal cation-exchange chromatography) and thus of two different adsorption modes are paramount. These allow, for example, the binding of washed-out protein A or contaminants which coelute with the antibody from protein A on the multimodal matrix, whereas the antibody remains in the mobile phase or can be eluted separately from the contaminants. Strictly speaking, the loading capacity of the protein A column cannot be utilized more efficiently in this method, since (i) a sensor for detecting the breakthrough following the affinity-chromatographic step is missing, (ii) the flow-through during loading of the affinity-chromatographic column is not guided onto the multimodal matrix and (iii) the eluate of the affinity-chromatographic step is loaded onto the multimodal matrix. Therefore, savings in the capacity of the affinity-chromatographic matrix are barely possible with this method.
When loading a chromatographic column with the feedstream containing a target molecule, the loading operation is frequently prematurely terminated in order to avoid an overloading of the column and thus a loss of target molecule. The available capacity of the column is therefore not fully utilized. Especially in the case of expensive column materials, such as protein A for example, the result is an economically disadvantageous situation. Furthermore, the number of DSP cycles per batch or throughput is generally defined by the size of the available column material. Therefore, a longer total duration and a reduced efficiency of the process can arise as a further consequence.